The basilar papilla (BP) is the auditory organ of the chicken and houses sensory cells, known as hair cells, that are innervated by afferent and efferent neurons. Along the radial axis of the BP, the organization of hair cells and the pattern their neural innervation is such that on one side of the BP hair cells take on a tall morphology (tall hair cells) and are primarily innervated by afferents. On the other side of the BP hair cells take on a short morphology (short hair cells) and are primarily innervated by efferents. While many studies have examined the effects of axon guidance factors on innervation of the BP, it is currently unknown what factors drive this particular innervation pattern. Previous work has shown that Wnt9a overexpression in the developing BP results in an increase in hair cells that take on tall hair cell morphology and afferent innervation of those hair cells. RNA deep sequencing of control and Wnt9a overexpressing BPs has identified axon guidance genes, among other genes, that are downstream of Wnt9a and may be mediating the observed changes. This study proposes to examine the impact of three of these genes on neural innervation. The first and second aim will examine the role of Semaphorin-3F (Sema3F) and Contactin-6 (Cntn6), respectively, on the radial pattern of innervation. The third aim will examine the effects of Slit2 on Roundabout (Robo)-mediated activated ?-catenin (pY489-?-catenin) in the BP and statoacoustic ganglion (SAG). In these aims, in-situ hybridization will be used to determine the endogenous spatial and temporal expression pattern of these transcripts in cochlear ducts. In vitro and in vivo methods will be used to examine the effect of these genes on innervation. For in vitro experiments, SAGs will be cultured in the presence of purified axon guidance protein. For in vivo experiments, axon guidance genes will be overexpressed. Immunohistochemistry will be used to label neurites, hair cells, and downstream factors to detect responses to these manipulations. In Slit2 experiments, a TCF transcription factor reporter will be electroporated into the BPs to determine if pY489-?-catenin functions to activate transcription. The results from these experiments will provide insights into innervation of the BP. Genes are able to influence the radial pattern of innervation may be further studied for their ability to regenerate innervation in hair cells that have been damaged or destroyed. This knowledge will be foundational for the development of molecular therapies to treat hearing loss. This proposal will additionally provide training opportunities for the applicant in techniques such as transmission electron microscopy and in vitro culturing techniques, as well as experience in data quantification, data analysis, and scientific communication.